The combination of chemical separation and detection has long been recognized as invaluable to the analytical chemist in identifying chemicals at extremely low concentrations. One of the most successful combinations employs gas chromatography for separation and infrared spectroscopy for detection (P. R. Griffiths, J. A. De Haseth, Fourier Transform Infrared Spectrometry, Wiley InterScience, 481 (2007), ISBN 0470106298).
Recently, the combination of chromatography with surface-enhanced Raman spectroscopy (SERS) has been investigated to analyze trace chemicals in solution, at part-per-billion concentrations. Known advantages provided by SERS include high chemical specificity through abundant molecular vibrational information (similar to infrared spectroscopy), and extreme sensitivity, as demonstrated by the detection of single molecules. (See K. Kneipp, Y. Wang, R. R. Dasari and M. S. Feld, Appl. Spectrosc., 49, 780 (1995); and S. Nie and S. R. Emory, Science, 275, 1102 (1997)).
Initially, thin layer chromatography (TLC) was investigated with SERS. In one example, the TLC plates were used to first separate the analytes, were then coated with a silver colloid to generate SERS, and finally were employed to make measurements using a Raman spectrometer (J-M. L. Sequaris and E. Koglin, Anal. Chem., 59, 525 (1987)); the entire analysis took from 30 to 60 minutes. In a second example, the TLC plates were coated with silver prior to attempted separation but, as the authors conclude (N. J. Szabo and I. D. Winefordner, Appl. Spectrosc., 51, 965 (1997), separation required 40 to 45 minutes, SERS activity was poor, and “Neither material [TLC plates] was found to be suitable for this application [i.e., TLC plus SERS].” In a similar example (L. M. Cabalin and J. J. Laserna, Anal. Chim. Acta, 310, 337 (1995)), silver-coated filter paper was used to detect analytes by SERS. The analyte was added dropwise (spotted) onto the paper and dried; additional drops of analyte were added to the spot and dried, multiple times, and then the spot was measured using a two-dimensional detector.
To a large extent, liquid chromatography has replaced TLC for chemical separations, primarily because it is more efficient for separating chemicals. Separation efficiency, defined as the ability to spatially resolve chemicals in a relatively short period of time, typically 20 to 30 minutes, can be quantified in terms of the number of theoretical plates a column provides (L. S. Ettre, “Nomenclature For Chromatography”, Pure & Appl. Chem., 65, 819 (sp. p. 484) (1993)). The idea is that the column contains a number of separate hypothetical zones, stages or layers (i.e. theoretical plates), and that the analyte comes to equilibrium between the stationary phase and the mobile phase at each layer as the mobile phase moves down the column. Knowing the retention time, tR, and the width at half height, W1/2, for the peak of an eluted analyte, the theoretical number of plates, Neff, can be determined for a conventional chromatography column according to the expression:Neff=5.545(tR)2/(W1/2)2 It is highly desirable to provide a large number of theoretical plates, viz: Neff>1000, because doing so normally results in a commensurately high separation efficiency.
In conjunction with combining LC with SERS, several researchers have developed methods to perform SERS measurements in flowing sample cells (G. T Taylor, S. K. Sharma, and K. Mohanan, Appl. Spectrosc., 44, 635 (1990); F. Ni, R. Sheng, and T. M. Cotton, Anal. Chem., 62, 1958 (1990); N. J. Pothier and R. K. Force, Appl. Spectrosc., 46, 147 (1992)). The first two research groups identified employed tubing to combine the flow of the analyte with SER-active silver colloids, prior to passing a SER measurement sample cell. This flow injection analysis method provided the bases for developing SERS as a detector for LC. In all cases except one (see below), standard LC columns were employed to first separate the analytes; flow injection analysis was then used to combine the column elute with silver colloids, the mixture of which was measured in a variety of flow-through sample cells (see R. D. Freeman, R. M. Hanmaker, C. E. Meloan, and W. G. Fateley, Appl. Spectrosc., 42, 456 (1988); R. Sheng, F. Ni and T. M. Cotton, Anal. Chem., 63, 437 (1991); L. M. Cabalin, A. Ruperez and J. J. Laserna, Talanta, 40, 1741 (1993); L. M. Cabalin, A. Ruperez and J. J. Laserna, Anal. Chim. Acta, 318, 203 (1996); and B. J. Kennedy, R. Milofsky and K. T. Carron, Anal. Chem., 69, 4708 (1997)); while in one case the eluted analytes were deposited as a series of drops onto a SER-active TLC plate, then measured (S. A. Soper, K. L. Ratzlaff, and T. Kuwana, Anal. Chem., 62, 1438 (1990)). In two cases, the authors report the separation efficiency in terms of theoretical plates. Sheng et al. demonstrate that four purine bases can be separated in 10 to 15 minutes, while Cabalin et al. (1993) demonstrate that three drugs can be separated in 8 to 10 minutes. The latter-named authors also quantified the separation efficiency in terms of theoretical plates, with Neff ranging from 860 to 2000 being deemed sufficient for analysis.
Previous research has employed primarily the three most common methods of generating surface-enhanced Raman scattered radiation; i.e., using roughened silver or gold electrodes, using silver- or gold-coated substrates, and using silver or gold colloids for detecting separated analytes. The lattermost method has gained the greatest amount of attention, since colloids can be prepared easily and inexpensively, and mixing of the colloids with the chromatographic column effluent, using flow injection, is straightforward. Care must be taken however to control aggregation of the colloids so that the amount of Raman signal enhancement is maintained. Also, a range of experimental variables, such as analyte concentration and pH, can strongly influence aggregation and, to some extent, limit applications; the choice of carrier solvent is similarly limited by the need to maintain colloid integrity.
As described by Farquharson et al. in commonly owned U.S. Pat. No. 6,623,977 (filed under application Ser. No. 09/704,818, and published as International Publication No. WO 01/33189 A2), the entire specification of which is hereby incorporated hereinto by reference thereto, sol-gels have been developed to trap silver or gold particles as an improved method of generating plasmons for SERS (see also S. Farquharson, P. Maksymiuk, K. Ong and S. D. Christesen, SPIE, 4577, 166 (2002); F. Akbarian, B. S. Dunn and J. I. Zink, J. Chem. Phys., 99, 3892 (1995); T. Murphy, H. Schmidt and H. D. Kronfeldt. SPIE, 3105, 40 (1997); and Y. Lee, S. Dai and J. Young, J. Raman Spectrosc. 28, 635 (1997)). It is appreciated that, once the sol-gel has formed, the particle size and aggregation of the metal dopant are stabilized, albeit changes in pH may still result in variable Raman signal intensities, such as in the case of weak acids and bases, wherein the relative concentrations of the ionized and unionized forms may be influenced. Also, it has been shown that many of the common solvents, such as acetone, methanol, and water, can be used equally with these SER-active metal-doped sol-gels in generating SER spectra of analytes.
In accordance with other recent developments, moreover, sol-gels have been used as the stationary phase in columns for liquid- and gas-phase chromatography, affording advantages in both the preparation of columns and also in their performance. The sol-gel approach enables deactivation, coating, and immobilization to be combined as a single step, while the sol-gels have found broader application to solvents and analytes.
Microchip devices have also been employed for effecting chemical separations (see S. C. Jacobson, R. Hergenröder, L. B. Koutny, and J. M. Ramsey, Anal. Chem., 66, 1114 (1994); S. C. Jacobson, R. Hergenröder, L. B. Koutny, R. J. Warmack, and J. M. Ramsey, Anal. Chem., 66, 1107-1113 (1994); S. C. Jacobson, R. Hergenröder, L. B. Koutny, and J. M. Ramsey, Anal. Chem., 66, 2369 (1994); and A. W. Moore, Jr., S. C. Jacobson, and J. M. Ramsey, Anal. Chem., 67, 4184 (1995)).
It is clear from the foregoing background disclosure that chromatography and SERS can be combined to achieve trace chemical analysis of multiple analytes in a sample. It is also clear that separation materials used in TLC can be coated with silver to combine chemical separation and SER-activity, albeit the separation process is slow (sp. 40 to 45 minutes), and the SER-activity is modest. Furthermore, the foregoing disclosure indicates that modest improvements in analysis time (sp. 10 minutes) and SER-activity can be made by adding silver colloids to the effluent of liquid chromatography columns.
No one skilled in the art of TLC or LC suggests using transparent containment means to hold a material that has the combined abilities of performing chemical separation and exhibiting SER-activity, so that Raman spectroscopy can be used to make measurements at discrete points or continuously along the length of the containment means, as this would not provide any advantage over the TLC or LC methods described. Knowledge of the prior art would indicate that, in the latter case (i.e., LC), the Raman measurement would still be best performed at the end of the column, where efficient separation has been achieved, as defined by a high number of theoretical plates. Indeed, Nirode et al. (W. F. Nirode, G. L. Devault, and M. J. Sepaniak, Anal. Chem., 72, 1866 (2000)) describe measuring SER spectra of separated analytes as they flow by the end of an electrophoresis column. In that method, silver colloids were added to a running buffer containing the mixed analytes, which flowed through a capillary connecting the anode and cathode of an electrochemical cell. Although measurements along the length of the column might conceivably have been performed (if the structurally supporting coating were etched away at several positions, thereby most likely rendering the column too fragile to be of use), analyte separation prior to the column exit point is incomplete; and more importantly, the analytes are moving, making it implausible to know where along the column, or when, to make such measurements. The same would be true for LC, and thus no advantage would be expected.
In no case does the foregoing background information teach or remotely suggest rapidly drawing a sample into a transparent column, capillary, or channel that contains a combined chemical separation and SER-active medium, to afford effective distribution and rapid separation of chemical analytes along the length of such containment means, so that the analytes can be quickly detected and measured by Raman spectroscopy at a plurality of points along the length of the containment means. One skilled in the art of chromatography would not expect to achieve the extraordinary level of detection and discrimination capability that is afforded by the present method and apparatus, based for example upon a characterization of the invention in terms of the number of theoretical plates presented for analyte chemical separation to allow identification of the analytes.